Peptide hydrolyzing enzymes (peptidases) catalyze the biosynthesis and inactivation of neuropeptides. Elucidation of the properties of these enzymes is of fundamental importance in the study of neuropeptide function. A broad based approach combining biochemistry, medicinal chemistry, molecular biology, immunohistochemistry and physiology to the study of the biological significance of thyrotropin releasing hormone (TRH) and angiotensin metabolizing enzymes is proposed. Neuronally released TRH is degraded by pyroglutamyl peptidase II (PP II), a synaptosomal membrane- bound ectoenzyme. PP II is predominantly found in brain and is the only known neuropeptide-specific peptidase. It is subject to multiple modes of regulation including thyroid hormone up-regulation and a protein kinase C mediated rapid down-regulation. Studies on PP II will be continued by cloning the rat brain enzyme and determining the tissue distribution of its mRNA. The molecular mechanisms of catalysis and regulation will be explored by site directed mutagenesis. Improved inhibitors will be synthesized to probe its physiological significance. Such inhibitors may provide an indirect approach to the potentiation of the biological activity of exogenously administered or endogenous TRH. All of the components of the renin-angiotensin (ang) system are present in brain. It has been proposed that part or all of the biological activity of the brain renin-ang system is mediated by ang III (des Asp-ang II). The conversion of ang II to ang III is catalyzed by the well characterized glutamyl aminopeptidase (aminopeptidase A). A new assay uncovered the presence of a second enzyme in brain, aspartyl aminopeptidase (D-AP), also catalyzing this biotransformation. To determine the relative significance of these enzymes in the brain renin-ang system, rat brain D-AP will be purified to homogeneity and cloned. The substrate specificities of both enzymes will compared to facilitate the design of specific inhibitors. The properties of the inhibitors will be evaluated in collaborative studies. Neuropeptide degrading enzymes represent attractive targets of pharmacological intervention. Modification of endogenous neuropeptide function by inhibition of metabolism represents an approach to the therapy of neurological disorders.